Abstract
Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a ligand of low-density lipoprotein (LDL) receptor (LDLR) that promotes LDLR degradation in late endosomes/lysosomes. In human plasma, 30–40% of PCSK9 is bound to LDL particles; however, the physiological significance of this interaction remains unknown. LDL binding in vitro requires a disordered N-terminal region in PCSK9's prodomain. Here, we report that peptides corresponding to a predicted amphipathic α-helix in the prodomain N terminus adopt helical structure in a membrane-mimetic environment. This effect was greatly enhanced by an R46L substitution representing an atheroprotective PCSK9 loss-of-function mutation. A helix-disrupting proline substitution within the putative α-helical motif in full-length PCSK9 lowered LDL binding affinity >5-fold. Modeling studies suggested that the transient α-helix aligns multiple polar residues to interact with positively charged residues in the C-terminal domain. Gain-of-function PCSK9 mutations associated with familial hypercholesterolemia (FH) and clustered at the predicted interdomain interface (R469W, R496W, and F515L) inhibited LDL binding, which was completely abolished in the case of the R496W variant. These findings shed light on allosteric conformational changes in PCSK9 required for high-affinity binding to LDL particles. Moreover, the initial identification of FH-associated mutations that diminish PCSK9's ability to bind LDL reported here supports the notion that PCSK9-LDL association in the circulation inhibits PCSK9 activity.
Highlights
Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a ligand of low-density lipoprotein (LDL) receptor (LDLR) that promotes LDLR degradation in late endosomes/lysosomes
Gain-of-function PCSK9 mutations associated with familial hypercholesterolemia (FH) and clustered at the predicted interdomain interface (R469W, R496W, and F515L) inhibited LDL binding, which was completely abolished in the case of the R496W variant
We previously reported that association of PCSK9 with LDL particles in vitro depends on an intrinsically disordered region (IDR) in the prodomain N terminus [18]
Summary
To test the influence of peptide backbone conformation on LDL binding, we introduced proline residues at two separate positions in the putative helix (L41P and A44P) in full-length recombinant PCSK9 (Fig. 4A). Proline substitution inhibited the ability of secreted PCSK9 to bind to LDL by Ͼ90% (Fig. 4B), supporting an important structural role of helical conformation in the prodomain N terminus. When added to the medium of cultured HepG2 cells, PCSK9-A44P was able to mediate dose-dependent degradation of cell surface LDLRs at levels comparable with WT PCSK9 (Fig. 5D), in agreement with a recent study [29] These data suggest that helical conformation in the prodomain N terminus plays a larger role in LDL binding relative to its role in PCSK9-LDLR association and LDLR degradation. Mean Residue Ellipticity (deg cm dmol-1) x 10-3 Mean Residue Ellipticity (deg cm dmol-1) x 10-3
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